TRIP13通过同源重组通路提高肺腺癌细胞的放射抗性

背景与目的 放疗是非小细胞肺癌（non-small cell lung cancer, NSCLC）最常用的治疗手段之一。然而，一部分肿瘤细胞对放射线的不敏感是放疗疗效差、患者预后不良的重要原因之一，探究放射抵抗背后的深层机制是解决这一临床难题的关键。本研究旨在寻找与肺腺癌（lung adenocarcinoma, LUAD）放射抵抗相关的分子，初步经数据库筛选锁定甲状腺素受体结合因子13（thyroid hormone receptor interactor 13, TRIP13）为主要研究对象，并探索TRIP13是否与LUAD的放射抵抗有关及具体机制，以期为临床接受放疗的LUAD患者的联合治疗提供理论依据和潜在靶点。方法 选取基因表达综合数据库（Gene Expression Omnibus, GEO）中的GSE18842、GSE19188和GSE33532共3个数据集，借助R 4.1.3软件分别筛选3个数据集中差异表达的基因（|log FC|>1.5, P<0.05），之后使用Venn diagram找出在3个数据集中共有的差异表达基因。随后，借助STRING在线工具和Cytoscape软件，对筛选出来的差异基因进行蛋白质相互作用分析和模块分析，借助Kaplan-Meier Plotter数据库对各基因进行生存预后分析，并确定TRIP13基因作为后续主要研究分子。随后，采用亚致死性剂量照射法对人LUAD细胞系H292进行多次X射线照射，以构建具有放射抗性的细胞系H292DR。采用细胞计数试剂盒-8（cell counting kit-8, CCK-8）实验和克隆形成实验验证H292DR细胞的放射抗性能力。Western blot检测H292细胞和H292DR细胞中TRIP13蛋白的表达水平。使用小干扰RNA（small interfering RNA, siRNA）沉默H292DR细胞中TRIP13蛋白的表达并进行Western blot检测。观察TRIP13沉默后H292DR细胞的克隆形成能力和迁移能力，随后检测共济失调-毛细血管扩张突变（ataxia telangiectasia mutated, ATM）蛋白等与同源重组密切相关的蛋白的表达水平变化。 结果 经多个GEO数据集筛选、外部数据集的验证以及生存分析发现，TRIP13在LUAD中高表达，并与接受过放疗的LUAD患者的不良预后有关；并且，TRIP13基因富集分析（gene set enrichment analysis, GSEA）的结果提示，TRIP13可能通过促进放疗后的同源重组修复而与LUAD放射抵抗有密切关联。经实验检测发现，TRIP13的表达在H292DR中上调，而沉默TRIP13后能够增加H292DR细胞对放射线的敏感性。 结论 TRIP13与接受放疗后的LUAD患者的预后不良有关，可能是通过促进同源重组修复途径来介导LUAD细胞对放射线的抵抗。

•2• 中 国 肺 癌 杂 志 2 0 2 4 年 月 第 2 7 卷 第 期 C h i n J L u ng C a nc er, Ja nu a r y 2 0 2 4 , Vol .27, No.   enocarcinoma (LUAD), identified thyroid hormone receptor interactor 3 (TRIP3) as the main target initially, and explored whether TRIP3 is related to radioresistance in LUAD and the specific mechanism, with the aim of providing theoretical basis and potential targets for the combination therapy of LUAD patients receiving radiotherapy in the clinic.Methods Three datasets, GSE8842, GSE988 and GSE33532, were selected from the Gene Expression Omnibus (GEO) database and screened for differentially expressed genes (|log FC|>.5, P<0.05) in each of the three datasets using the R 4..3 software, and then Venn diagram was used to find out the differentially expressed genes common to the three datasets.The screened differential genes were then subjected to protein-protein interaction (PPI) analysis and module analysis with the help of STRING online tool and Cytoscape software, and survival prognosis analysis was performed for each gene with the help of Kaplan-Meier Plotter database, and the TRIP13 gene was identified as the main molecule for subsequent studies.Subsequently, the human LUAD cell line H292 was irradiated with multiple X-rays using a sub-lethal dose irradiation method to construct a radioresistant cell line, H292DR.The radioresistance of H292DR cells was verified using cell counting kit-8 (CCK-8) assay and clone formation assay.The expression levels of TRIP3 in H292 and H292DR cells were measured by Western blot.Small interfering RNA (siRNA) was used to silence the expression of TRIP3 in H292DR cells and Western blot assay was performed.The clone formation ability and migration ability of H292DR cells were observed after TRIP3 silencing, followed by the detection of changes in the expression levels of proteins closely related to homologous recombination, such as ataxia telangiectasia mutated (ATM) protein.Results Screening of multiple GEO datasets, validation of external datasets and survival analysis revealed that TRIP3 was highly expressed in LUAD and was associated with poor prognosis in LUAD patients who had received radiation therapy.And the results of gene set enrichment analysis (GSEA) of TRIP13 suggested that TRIP3 might be closely associated with LUAD radioresistance by promoting homologous recombination repair after radiation therapy.Experimentally, TRIP3 expression was found to be upregulated in H292DR, and silencing of TRIP3 was able to increase the sensitivity of H292DR

Fig 3 TRIP3可能会促进放疗后的HR
Fig 3 Relationship between TRIP13 expression and survival prognosis of lung cancer patients.A: Significant correlation between TRIP13 expression and survival prognosis of lung cancer patients (n=65) treated with radiotherapy as shown in the Kaplan-Meier Plotter database; B, C: Dual demonstration of no significant correlation between TRIP13 and survival prognosis of patients with LUSC as shown in the Kaplan-MeierPlotter database (n=524) and the GSE37745 dataset (n=66).HR: hazard ratio; LUSC: lung squamous cell carcinoma.

Fig 6 Fig 7 Fig 8
Fig 6 Construction and validation of radioresistant LUAD cell lines and detection of TRIP13 protein expression.A, B: Clone formation assay was used to verify the radioresistant phenotype of H292DR, and survival curves were plotted; C: CCK-8 cell proliferation assay was performed to verify the radioresistant phenotype of H292DR; D: Observation of H292 and H292DR cells under bright-field of view of microscope (×40); E: Western blot was used to detect the TRIP13 protein expression in H292 and H292DR cells.OD: optical density; IR: radiation resistance; CCK-8: cell counting Kit-8.**P<0.01,****P<0.0001.
cells to radiation.Conclusion TRIP3 is associated with poor prognosis in LUAD patients treated with radiation, possibly by promoting a homologous recombination repair pathway to mediate resistance of LUAD cells to radiation.
【Key words】 Lung neoplasms; Radioresistance; TRIP3 protein; Homologous recombination 【Copyright statement】Copyright © 2024, Chinese Journal of Lung Cancer.This study was supported by the grant from the China-Japan Friendship Hospital Talent Introduction Research Initiation Fund (No.206-RC-4)(to Guangying ZHU).
Univariate and multivariate Cox regression analyses of prognostic factors in 65 patients with LUAD who had received radiotherapy in the